Targeting a truncated Ho-endonuclease of yeast to novel DNA sites with foreign zinc fingers.
Ho-endonuclease of the yeast, Saccharomyces cerevisiae, initiates a mating type switch by making a site-specific double strand break in the mating type gene, MAT. Ho is a dodecamer endonuclease and shares six of the seven intein motifs with PI- Sce I endonuclease, an intein encoded by the VMAI gene. We show that a 113 residue truncated Ho-endonuclease starting at intein motif C initiates a mating type switch in yeast. Ho is the only dodecamer endonuclease with zinc fingers. To see whether they have a role in determining site specificity we exchanged them for zinc fingers of the yeast transcription factor, Swi5. A chimeric endonuclease comprising the dodecamer motifs of Ho (C-E) and the zinc finger domain of Swi5 cleaves a Swi5 substrate plasmid in vivo. A similar chimera with the zinc fingers of SpI cleaves a GC box rich substrate plasmid. These experiments delineate a catalytic fragment of Ho-endonuclease that can be fused to various DNA binding moieties in the design of chimeric endonucleases with new site specificities.
Nahon
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Department of Life Sciences, Ben Gurion University of the Negev, Beersheba 84105, Israel.
Raveh
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D000595:Amino Acid Sequence; D001665:Binding Sites; D018797:Cell Cycle Proteins; D004271:DNA, Fungal / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005800:Genes, Fungal; D049770:Genes, Mating Type, Fungal; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D013379:Substrate Specificity; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016335:Zinc Fingers / Q000235:genetics
D018797:Cell Cycle Proteins; D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D011993:Recombinant Fusion Proteins; C080218:SWI5 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; C530845:HO protein, S cerevisiae; C037725:SCEI protein, S cerevisiae; D015252:Deoxyribonucleases, Type II Site-Specific